N-butyl-N-[(triethoxysilyl)methyl]butan-1-amine

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

yes

Remarks:

The deviations did not influence the quality or integrity of the present study.

GLP compliance:

yes (incl. QA statement)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The liquid test item was applied undiluted. 30 µL of the test item were dispensed directly atop the EpiDerm(TM) tissue. Afterwards, a nylon mesh was used as spreading tool and carefully placed on the tissue surface. The compatibility of the test item with the nylon mesh was checked in a pre-test.

Details on study design:

Test System:
The test was carried out with the reconstituted three-dimensional human skin model EpiDerm(TM) (MatTek). This skin model consists of normal (non-cancerous), human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts (Millicell(R)). The EpiDerm(TM) skin model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous, granular and cornified layers analogous to those found in vivo.

Provided Materials:
The EpiDerm(TM) tissues were provided as kits (EPI-200-SIT, MatTek), consisting of the following components relevant for this study:
1x sealed 24-well plate containing 24 inserts with tissues on Agarose (Lot: 19684 Kit C)
2x 24-well plates
8x 6-well plates
1x bottle of assay medium (DMEM-based medium, Lot: 100914 TMD)
1x bottle of DPBS Rinse Solution
1x 1 vial 5% SDS Solution (TC-SDS-5%)
25 pieces Nylon Mesh circles (8 mm diameter, 200 µm pore)
For MTT reduction assay the MTT assay kit (MTT-100; MatTek) was provided, consisting of:
1 vial MTT concentrate (Lot 092314MHA)
1 vial MTT diluent (supplemented DMEM; Lot 1553587)
1 bottle extractant solution (Isopropanol; Lot 072914ZSA)
 
Pre-Experiments:
To check the MTT-reducing capability of the test item 30 µL of the test item were mixed per 1 mL MTT medium and incubated for 1 h in an incubator at 37 ± 1 °C. If the mixture turns blue/purple, the test item is presumed to have reduced MTT.
To check the colouring potential of the test item 30 µL of the test item were mixed per 300 µL aqua dest. in a transparent recipient and incubated at 37 ± 1°C for 60 min.
Liquid test item was tested for mesh compatibility by applying 30 µL of the test item to a nylon mesh, placed on a slide. After 60 min. the mesh was examined microscopically. If the test item interacts with the mesh, it was applied without mesh as spreading aid.

Experimental Procedure:
Upon receipt of the EpiDerm(TM), the tissues were transferred into 6-well plates containing 0.9 mL pre-warmed assay medium per well. The 6-well plates were incubated overnight in a humidified incubator at 37 ± 1 °C, 5.0% CO2.
After this pre-incubation the tissues were treated with each dose group in triplicate, starting with the negative control. Start time was recorded with dosing of the first tissue staggered in one-minute intervals. After dosing of all tissues, all plates were transferred to the incubator for 35 ± 1 min. After 35 min, all plates were removed from the incubator and placed under the sterile flow until the 60 ± 1 min incubation time of the first dosed tissue were over. After 60 ± 1 min the tissues were washed intensively with DPBS, staggered in one-minute intervals. Excess DPBS was removed by blotting the bottom with blotting paper. The inserts were placed in a prepared 6-well plate containing 0.9 mL pre-warmed fresh assay medium and post-incubated at 37 ± 1 °C, 5.0% CO2, humidified to 95% for 24 ± 2 h. Following this incubation the tissues were transferred to new medium and incubated for additional 18 ± 2 h.
After this incubation period the inserts were transferred in a prepared 24-well plate containing 300 µL pre-warmed MTT medium and further incubated for 3 h ± 5 min at 37 ± 1 °C, 5.0% CO2, humidified to 95%.
After the 3 h ± 5 min MTT incubation period the tissues were rinsed three times with DPBS and afterwards placed on blotting paper to dry the tissues. The tissues were immersed in 2 mL isopropanol, sealed to inhibit evaporation and incubated at room temperature for at least two hours. At the end of the formazan extraction period the plate was mixed by shaking until the solution colour became homogeneous. Per each tissue 2 x 100 µL aliquots of the extract were mixed with 100 µL isopropanol and transferred into a 96-well plate and OD was measured at 550 nm without reference wavelength in a plate spectrophotometer.
 
Data Analysis:
Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with DPBS. The test item is considered to be irritant to skin in accordance with regulation EC 1272/2008 (UN GHS Category 2), if the tissue viability after 15 min of exposure and 42 h of post-incubation is less or equal to 50%. The test substance may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is higher than 50%.

Mean tissue viability Prediction I / NI
(% negative control)
≤ 50 % Irritant (I): UN GHS Category 2
> 50 % Non-Irritant (NI): UN GHS No Category

Test Acceptance Criteria:
The test meets acceptance criteria if:
- mean OD550 nm of the three negative control tissues is ≥ 0.8 and ≤ 2.8
- mean relative tissue viability of the three positive control tissues is ≤ 20%
- standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is < 18%.

Irritation / corrosion parameter:

other: other: relative tissue viability

Value:

62.8

Remarks on result:

other:

Remarks:

Basis: other: mean optical density. Time point: After 60 min exposure and 42 h post incubation period . Max. score: 100.0. (migrated information)

Irritant / corrosive response data:

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (62.8%) after 60 min treatment and 42 h post incubation.

The controls confirmed the validity of the study. The mean absolute OD550 of the three negative control tissues was ≥ 1 and ≤ 2.5. The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (2.7%). The maximum standard deviation of viability of replicate tissues of all dose groups was < 18% (0.2% - 5.4%).

Other effects:

The mixture of 30 µL test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple.
The mixture of 30 µL / 25 mg of the test item per 300 µl aqua dest. showed no colouring detectable by unaided eye-assessment.
Microscopical examination of a nylon mesh treated for 60 min. with 30 µL of the test item showed no interactions. Therefore, the nylon mesh was used as spreading aid.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In this study under the given conditions the test item showed no irritant effects. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

Executive summary:

SUMMARY

In the present study the skin irritant potential of N,N-Dibutylaminomethyl-triethoxysilan was analysed.The EpiDerm™-Standard Model (EPI-200™), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404) to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a60 min exposure and 42 h post incubation periodand compared to those of the concurrent negative controls.

In this study under the given conditions the test item showed no irritant effects. The relative mean tissue viability after 60 min of exposure and 42 h post incubation was> 50%. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Species/strain: healthy New Zealand White Rabbits, Crl: KBL (NZW)
- Source: Charles River Deutschland, 97633 Sulzfeld, Germany
- Sex: female
- Body weight at the beginning of the study: > 2 kg
- Age at the beginning of the study: animals no. 1 and no. 2: 31 weeks old animals no. 3: 19 weeks old
- Number of animals: 3

The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes.

ENVIRONMENTAL CONDITIONS
- Semi barrier in an air-conditioned room
- Temperature: 18 ± 3 °C (recommendations of TVT, GV-SOLAS)
- Relative humidity: 55 ± 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: at least 10 x / hour
- Free access to autoclaved hay and to Altromin 2123 maintenance diet for rabbits (lot no. 0724), rich in crude fibre
- Free access to tap water (drinking water, municipal residue control, microbiological controls at regular intervals)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Housed in ABS-plastic or Noryl rabbit cages, floor 4200 cm2
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

A dose of 0.1 mL of the test item was applied to the test site.

Duration of treatment / exposure:

The test item was applied at a single dose in the conjunctival sac of one eye of each test animal after pulling the lower lid away from the eyeball. The lids were then gently held together for about 1 second in order to prevent loss of the material. The untreated contralateral eye served as control. The treated eye was not rinsed after the application.

Observation period (in vivo):

The animals were observed for 72 hours after dosing.

Number of animals or in vitro replicates:

3

Details on study design:

Preparation of the Animals:
Approximately 24 hours before the test and immediately prior to the application both eyes of each animal were examined. A health inspection was performed to ensure the good state of health of the animals. Approximately 24 hours before the application the eyes were also examined with the aid of a fluorescein solution (Fluoreszein SE Thilo, Alcon Pharma GmbH, lot no. H 401, expiry date: 04/2016). The eyes were rinsed with physiological saline 0.9% NaCl (B. Braun Melsungen, lot no. 1307034, expiry date: 06/2016) after the examination. None of the animals showed eye irritation, ocular defects, or pre-existing corneal injury.

Initial Test (In Vivo Eye Irritation/Corrosion Test Using One Animal):
The in vivo test was performed initially using one animal.

Application:
One hour before the application of the test item, 0.01 mg/kg of buprenorphine (Reckitt Benckiser, lot no. 5416, expiry date: 09/2016) was administered subcutaneously in order to achieve a therapeutic level of systemic analgesia. Approximately 5 minutes prior to the application of the test item, 2-3 drops of an ocular anaesthetic (proparacaine hydrochloride ophtalmic 0.5% solution, Ursapharm Arzneimittel, lot no. 282598, expiry date: 04/2016) were administered in both the treated and the control eye of each animal. The test item was applied at a single dose in the conjunctival sac of one eye of each test animal after pulling the lower lid away from the eyeball. The lids were then gently held together for about 1 second in order to prevent loss of the material. The untreated contralateral eye served as control. The treated eye was not rinsed after the application.

Confirmatory Test:
The results of the initial test did not indicate the test item to be corrosive or a severe irritant to the eye using the procedure described. In order to confirm the response, two additional animals were treated in the same manner.

Observation Period:
The animals were observed for 72 hours after dosing.

Clinical Observation:
The eyes were examined for signs of irritation throughout the observation period. The eye irritation was scored and recorded according to the grades in the table below. For the calculation only the 24, 48 and 72-hour readings were used. At the end of the observation period the treated eyes were examined with the aid of a fluorescein solution. The eyes were rinsed with physiological saline 0.9% NaCl after the examination.

Evaluation of Results:
Individual reactions for each animal were recorded according to the scoring system described in Table 2 at each time of observation. For the calculation only the 24, 48 and 72-hour readings were used. Nature, severity and duration of clinical observations were described. The body weight changes were summarised in a tabular form.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

other: 24, 48, and 72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Time point:

other: 24, 48, and 72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

other: 24, 48, and 72 h

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: 24, 48, and 72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

After the application into the eyes of three female NZW rabbits the test item produced neither irritant nor corrosive ocular effects.

Neither mortalities nor significant clinical signs of toxicity were observed.

Upon fluorescein examinations at the end of the observation period of 72 hours no corneal lesions were found in any animal.

Conjunctival discharge was observed in all animals 1 hour post-application.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of the present study, a single ocular application of the test item N,N-Dibutylaminomethyl-triethoxysilan to rabbits at a dose of 0.1 mL produced no irritant effects. Neither mortalities nor significant clinical signs of toxicity were observed.

In conformity with the EC criteria for classification and labelling requirements for dangerous substances and preparations according to Annex VI of Commission Directive 2001/59/EC, the test item N,N-Dibutylaminomethyl-triethoxysilan has no obligatory labelling requirement for eye irritation.

According to Annex I of Regulation (EC) 1272/2008, the test item N,N-Dibutylaminomethyl-triethoxysilan has no obligatory labelling requirement for eye irritation.

According to GHS (Globally Harmonized Classification System) the test item N,N-Dibutylaminomethyl-triethoxysilan has no obligatory labelling requirement for eye irritation.

Executive summary:

SUMMARY RESULTS

On the basis of the test results given below and in conformity with the criteria given in Annex VI to Commission Directive 2001/59/EC the substance should be assigned the following risk phrase:

No phrase

On the basis of the test results given below and in conformity with the criteria given in Annex I of Regulation (EC) 1272/2008, the substance should be:

Not classified

On the basis of the test results given below and in conformity with the criteria given in GHS (Globally Harmonized System of Classification and Labelling of Chemicals), the substance should be:

Not classified

Species/strain: New Zealand White Rabbits Crl: KBL (NZW)

Number of animals: 3

Amount of substance: 0.1 mL per test site

First time of effects: all animals: 1 hour post-application discharge grade 1

Last time of effects: all animals: 1 hour post-application discharge grade 1

Reversibility of the observed effects: all animals: the changes were fully reversible within 24 hours post-application

Method: OECD 405, EC 440/2008, Method B.4, OPPTS 870.2400

The calculated mean scores (Table 1) did not exceed the limit values according to Directive 2001/59/EC, to Regulation (EC) 1272/2008 and to GHS.

Table 1: Average Irritation Scores – (24, 48, 72-hour reading)

Animals No.	Gender	Conjunctival Redness	Conjunctival Chemosis	Iris	Cornea Opacity
1	Female	0.00	0.00	0.00	0.00
2	Female	0.00	0.00	0.00	0.00
3	Female	0.00	0.00	0.00	0.00

Conclusion:

Under the conditions of the present study, a single ocular application of the test item N,N-Dibutylaminomethyl-triethoxysilan to rabbits at a dose of 0.1 mL produced no irritant effects. Neither mortalities nor significant clinical signs of toxicity were observed.

In conformity with the EC criteria for classification and labelling requirements for dangerous substances and preparations according to Annex VI of Commission Directive 2001/59/EC, the test item N,N-Dibutylaminomethyl-triethoxysilan has no obligatory labelling requirement for eye irritation.

According to Annex I of Regulation (EC) 1272/2008, the test item N,N-Dibutylaminomethyl-triethoxysilan has no obligatory labelling requirement for eye irritation.

According to GHS (Globally Harmonized Classification System) the test item N,N-Dibutylaminomethyl-triethoxysilan has no obligatory labelling requirement for eye irritation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Two acute dermal Irritation in vitro studies (EpiSkin, EpiDerm) according to OECD 439 with N,N-Dibutylaminomethyl-triethoxysilane was performed.

The test substance was considered as non-irritant to skin in both tests in accordance with UN GHS “No Category”, because the tissue viability after exposure and post-treatment incubation is higher than 50%.

Therefore, the substance does not have to be classified.

An acute eye Irritation/Corrosion in vitro study according to OECD 437 with N,N-Dibutylaminomethyl-triethoxysilane was performed. The result of the test was ambiguous, due to that, an additional study according to OECD 405 with N,N-Dibutylaminomethyl-triethoxysilane was performed.

The test item was applied to the lower conjunctival sac of one eye of 3 New Zealand White Rabbits, Crl: KBL (NZW) at a dose of 0.1 ml per application site. The untreated eye served as control. After the application into the eyes of three female NZW rabbits the test item produced neither irritant nor corrosive ocular effects. Neither mortalities nor significant clinical signs of toxicity were observed. Upon fluorescein examinations at the end of the observation period of 72 hours no corneal lesions were found in any animal. Conjunctival discharge was observed in all animals 1 hour post-application. Therefore, the substance does not have to be classified.

Justification for selection of skin irritation / corrosion endpoint:
The reliable GLP compliant OECD Guideline study was chosen.

Justification for selection of eye irritation endpoint:
The reliable GLP compliant OECD Guideline study was chosen.

Justification for classification or non-classification

Both, the eye irritation (in vivo) and the skin irritation (in vitro) studies did not exhibit any irritating potential that leads to classification. With regard to the low volatility of the test item the same can be assumed for respiratory irritation.